Functional evaluation of HIV/SIV Nef as superantigen.

It is speculated that a virus-encoded superantigen is involved in the pathogenesis of human and simian immunodeficiency virus infections and that the accessory protein Nef might be that superantigen. We are able to show, using a murine superantigen screening system, that Nef does not display features characteristic of a superantigen. Upon transfection into MHC class II expressing antigen-presenting cells, it is expressed, but fails to induce Vbeta-specific expansion of peripheral T lymphocytes, which is a characteristic feature of superantigens in mixed lymphocyte culture. Therefore, we cannot support the hypothesis that Nef is a superantigen. The observations in favor of that hypothesis must be explained by other mechanisms.

Functional analysis of the env open reading frame in human endogenous retrovirus IDDMK(1,2)22 encoding superantigen activity.

Mice harbor a family of endogenous retroviruses, the mouse mammary tumor viruses (MMTV), which encode superantigens. These superantigens are responsible for the deletion of T cells expressing certain Vbeta chains of the T-cell receptor in the thymus. Human T cells are able to recognize MMTV-encoded superantigens presented by human major histocompatibility complex class II-positive cells. Owing to this and to the similarity of the human and murine immune systems, it was speculated that human endogenous retroviruses might also code for superantigens. Recently, it was reported that a proviral clone (IDDMK(1,2)22) of the human endogenous retrovirus family HTDV/HERV-K encodes a superantigen. The putative superantigen gene was located within the env region of the virus. Stimulated by these findings, we amplified by PCR and cloned into eucaryotic expression vectors open reading frames (ORFs) which were identical or very similar to IDDMK(1,2)22. When we transfected these vectors into A20 cells, a murine B-cell lymphoma, we were able to demonstrate mRNA expression and protein production. However, we did not find any evidence that the ORF stimulated human or murine T cells in a Vbeta-specific fashion, the most prominent feature of superantigens.

Activation of macrophages and B lymphocytes by an oligodeoxynucleotide derived from an acutely pathogenic simian immunodeficiency virus.

Certain CpG-containing DNA sequences from bacteria, viruses, or invertebrates elicit responses in the vertebrate innate immune system. These responses also account for many nonspecific effects of oligodeoxynucleotides used for antisense approaches. Here we describe a sequence from an acutely pathogenic simian immunodeficiency virus (SIV) that induces release of cytokines from macrophages and B lymphocyte proliferation. Furthermore, several similar sequences in other immunodeficiency viruses were found that also activate macrophages. These results led to the question if CpG-containing DNA, which is thought to play an immunostimulatory role in bacterial infections, has a similar role in infections by immunodeficiency viruses.

The persistent variant of influenza C virus carries one characteristic point mutation in RNA segment 1.

Influenza C/ Ann Arbor/1/50-pi(C/AA-pi) virus causes persistent infections in MDCK and Wi38 cells, but sets limited, wild-type like infections in other cells. Concluding that persistence itself it dependent on the host environment, we determined the nucleotide sequence of the C/AA-pi analogous gene to the basic polymerase 2 (PB2) of influenza A virus, which is known to be a determinant for the host range. C/AA-pi and the parental wild-type virus (C/AA-wt) have 16 nucleotides in common that are different to a previously published PB2 sequence (C/JJ/50). These variations, which are probably due to divergent passages histories, are scattered along the sequence and are partially found in another published isolate (C/Berlin/1/85). One single mutation, however, is unique to the persistent variant. Nucleotide 28 mutated from T to C which leads to a change of amino acid 3 from Leu to Phe. This substitution is stably associated with the persistent phenotype throughout multiple passages in different cells lines and eggs and cannot be found in any other influenza C viruses.

Nucleotide-specific PCR for molecular virus typing.

Nucleotide sequence studies detected a double-point mutation in the genomic RNA segment 4 (nt 871 and 872) of the persistent variant C/AA-pi of influenza C/Ann Arbor/1/50 virus. The 3'-end-points of two distinct PCR primers were positioned exactly at this genome location and thereby adjusted the priming determinant complementary to the varied strain or to its wild-type counterpart. Consequently, positive RT-PCR products strictly referred to one of the two viruses examined, in both cases, using either virion or infected-cell RNA templates. Artificial virus mixtures could easily be distinguished by this method in a subsequent qualitative gel analysis. PCR annealing conditions and control reactions were optimized, for the monitoring of influenza virus isolates throughout multifold passages. Thus, sequence diversity in just two neighbouring nucleotides is sufficient to determine whether or not successful PCR amplification takes place, and this method can be used as a reliable means of virus strain differentiation.